Intron-mediated enhancement of gene expression

Most genes in eukaryotes (well, at least eukaryotes that are not Saccharomyces cerevisiae) possess introns, sequences that are transcribed by RNA polymerase II and subsequently spliced out from the primary transcript.  Introns have been the subject of tremendous interest since their discovery in the 1970’s, and have provided much insight (and grist for controversy) into subjects as disparate as junk DNA, the RNA World, and mechanisms of gene expression.  Among the still-unresolved matters today has to do with the timing of splicing – is it cotranscriptional* or does it occur after polII has released the transcript.

The case for co-transcriptional splicing has been built in part through numerous studies that reveal physical connections between splicing factors and the transcriptional complex; many (most) of these involve the so-called CTD (C-Terminal Domain) of RNA polymerase II.  (This recent review summarizes this emerging field.)  The general idea is that, owing to the association of splicing factors with the CTD of polII, they are able to bind the nascent transcript and initiate splicing before polII has completed the synthesis of the primary transcript.

Alternative polyadenylation and cancer

This is a follow-up of sorts to a previous essay on the subject of alternative polyadenylation.  In the previous report, I discussed some bioinformatics studies that suggested that the 3′ UTRs of mRNAs change, in bulk, in the course of development in mammals.  The implication of these results is that poly(A) site choice in mammals is regulated, with important functional consequences.

A more recent study by Mayr and Bartel adds to this notion.  These authors studied 3′ UTR length in normal and cancer cells, and found a striking correlation between 3′ UTR length and the expression of oncogenes.  Specifically, higher expression (as is found in cancer cells) is correlated with shorter 3′ UTR.  As 3′ UTR length is determined by the position of the poly(A) site along a transcript, this implicates alternative polyadenylation as one mechanism by which oncogene expression is activated.

On the functional significance of alternative splicing

Alternative splicing – the choice of different splice sites and/or exons in a primary transcript that possesses numerous exons and introns – is a widespread phenomenon.  With the advent of very sensitive as well as high-throughput techniques, it has proven possible to identify alternatively-spliced transcripts for many, perhaps a majority, perhaps all genes.  However, the very sensitivity of the techniques raise the interesting and important question of the functional significance of what is observed.  Thus, it is possible that much (most, all?) of the alternatively-spliced mRNA isoforms are the results of splicing errors.  (Some in the blogosphere are of the opinion that alternative splicing is mostly artifact.)  Accordingly, studies that speak to the functions of the products of alternative splicing are always of interest.

A recent study from Stephen Mount’s lab illustrates an excellent approach to this problem.  In this study, two different isoforms of a so-called SR protein (the Arabidopsis SR45 splicing factor) were studied.  These isoforms are encoded by different alternatively-spliced mRNAs, and differ by eight amino acids that correspond to one of two 3′ splice sites that are chosen in the course of pre-mRNA processing.  Loss-of-function mutant plants that do not make SR45 show a range of developmental phenotypes that affect flowers and roots.  Interestingly, when one isoform is expressed* in a loss-of-function mutant background, the flower phenotype is reversed but not the root phenotype.  Conversely, expression of the other isoform restores normal root growth but not flower morphology.  The bottom line is that the two SR45 isoforms have distinct functions.  Thus, at least in this case, alternative splicing has important roles in growth and development.

Alternative polyadenylation in development

One of the things that is an open book is the true scope and physiological relevance of alternative polyadenylation.  A recent report in PNAS stirs this pot a bit (even if it leaves things still very much up in the air).  Briefly, this group has analyzed various large-scale gene expression repositories – ESTs, SAGE, and microarray – and found a tantalizing possible progression of 3′-UTR length during development.  Specifically, it seems as if global (or average) 3′-UTR length increases during the course of embryogenesis.  This change in the length of 3′-UTRs seems to be due to differential poly(A) site choice.  As I said, very tantalizing.

The abstract and brief commentary follows after the fold.

Where polyadenylation, siRNAs, and DNA methylation meet

It has become more apparent in recent years that the different aspects of gene expression – transcription initiation, transcription elongation, mRNA capping, splicing, and polyadenylation, transport of the mRNA to the cytoplasm, translation, and mRNA quality control – are rather extensively interconnected.  One corollary is that the polyadenylation complex, through various of its subunits, plays roles in various of these other processes.  This has been established for the most parts in mammalian and yeast models, but some recent work in plants is adding new and important variation to this theme.

A most recent of such studies has appeared online on PNAS.  This study, from the lab of Caroline Dean, reveals that the polyadenylation factor subunit FY (a homolog of the yeast protein Pfs2), acting in concert with the flowering regulator FCA, plays a crucial role in chromatin modifications that regulate the expression of the FLC gene.  Interestingly, this effect is not limited to just the FLC gene. Rather, other genes that are silenced by small RNA-mediated DNA methylation also require FY for this silencing.  This provocative finding seems to place FY in some sort of proximity to the small RNA-guided DNA methylation machinery, and may have some relevance to many aspects of transcription and mRNA quality control.

The abstract and citation follows. As always, enjoy.

Convergent transcription, polyadenylation, RNA editing, and regulation

One of the mechanisms by which polyadenylation may contribute to the regulation of gene expression (on paper, at least) involves gene pairs that are situated near each other and transcribed convergently.  In these instances, polyadenylation and transcription termination need to occur to prevent the production of RNAs that are anti-sense to the two members of the convergently-transcribed gene pair.  Overlapping transcripts could lead to the formation of double-stranded RNAs that could in turn trigger regulatory mechanisms, resulting in altered accumulation of the corresponding transcripts.

It is in this vein that a recent study from Gordon Carmichael’s lab at the University of Connecticut is of interest.  Briefly, these authors report that the early-to-late transition in gene expression in cells infected with the mouse polyoma virus is accomplished (at least in part) by a reduction in polyadenylation efficiency of the primary transcript encoding the so-called late genes.  Interestingly enough, this reduction in polyadenylation efficiency seems to be due to A-to-I editing of the region around the polyadenylation signal.  This editing in turn may be traced to an overlap of the early and late transcripts, such that double-stranded RNAs (the substrate for the A-to-I editing complex) that include the late polyadenylation signal are produced and edited before pre-mRNA processing occurs.

SOC1 and the RNA Underworld

Earlier, I described studies of the so-called SOC1 and FUL genes of Arabidopsis, genes that when mutated in concert change the growth habit of the plant is most remarkable ways.  A report that just came up on Plant Cell Online links one of these genes with one of the mechanisms by which RNAs are turned over in the cell.  Briefly, this study reveals that SOC1 expression is subject to posttranscriptional control, and that this control is linked with a component of the machinery that mediates nonsense-mediated decay (NMD) in plants.  This finding may be of interest for a number of reasons.  One is that NMD hasn’t yet been linked with lots of regulation in plants – it occurs, and we may infer conceptual links between alternative RNA processing and NMD, but much remains to be learned.  A second is that SOC1 functioning, previously implicated in important macroevolutionary transitions in plants, may be altered by many evolutionary processes, including those that affect RNA levels through NMD.

The abstract:

SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1) is regulated by a complex transcriptional regulatory network that allows for the integration of multiple floral regulatory inputs from photoperiods, gibberellin, and FLOWERING LOCUS C. However, the posttranscriptional regulation of SOC1 has not been explored. Here, we report that EARLY FLOWERING9 (ELF9), an Arabidopsis thaliana RNA binding protein, directly targets the SOC1 transcript and reduces SOC1 mRNA levels, possibly through a nonsense-mediated mRNA decay (NMD) mechanism, which leads to the degradation of abnormal transcripts with premature translation termination codons (PTCs). The fully spliced SOC1 transcript is upregulated in elf9 mutants as well as in mutants of NMD core components. Furthermore, a partially spliced SOC1 transcript containing a PTC is upregulated more significantly than the fully spliced transcript in elf9 in an ecotype-dependent manner. A Myc-tagged ELF9 protein (MycELF9) directly binds to the partially spliced SOC1 transcript. Previously known NMD target transcripts of Arabidopsis are also upregulated in elf9 and recognized directly by MycELF9. SOC1 transcript levels are also increased by the inhibition of translational activity of the ribosome. Thus, the SOC1 transcript is one of the direct targets of ELF9, which appears to be involved in NMD-dependent mRNA quality control in Arabidopsis.

The citation (hopefully, I will remember to update it once the paper comes out in print with the updated link for the paper copy):

Hae-Ryong Song, Ju-Dong Song, Jung-Nam Cho, Richard M. Amasino, Bosl Noh,  and Yoo-Sun Noh. The RNA Binding Protein ELF9 Directly Reduces SUPPRESSOR OF OVEREXPRESSION OF CO1 Transcript Levels in Arabidopsis, Possibly via Nonsense-Mediated mRNA Decay.  Plant Cell Advance Online Publication, Published on April 17, 2009; 10.1105/tpc.108.064774

More strangeness …

Awhile ago, I discussed a flurry of papers in Science that showed some curious aspects of transcription and promoters.  It seems as if every passing day brings a new report that pertains to the phenomenon.  A recent issue of Nature brings us two papers, back to back, that are relevant.  The bottom line is that bidirectional transcription is a widespread phenomenon, at least in yeast.  Moreover, this phenomenon is responsible, not just for divergent transcription of mRNA-encoding genes, but also for the production of so-called Cryptic Unstable Transcripts and other uncharacterized RNAs.  The abstracts and some brief commentary are beneath the fold.

Interesting stuff

While my yard is recovering from the ice, and I from today’s UK game, I thought I would toss out a few interesting abstracts that touch on important and contentious issues.  Peek beneath the fold and, as always, enjoy.

RNA and kitchen tools – Slicers, Dicers, and CRISPRs

RNA-based regulation is all the rage in biology today.  The more familiar mechanisms involve small RNAs such as microRNAs and silencing-associated RNAs.  The biogenesis and functioning of these RNAs involves enzymes and complexes that have been termed, among other things, Dicers and Slicers.  These subcellular kitchen utensils work by processing either the small RNA precursor or the base-paired target RNA.  This mode of regulation is most often associated with eukaryotes, and indeed homologous enzymes and mechanisms are not found in prokaryotes. However, systems with remarkable functional similarity may occur in bacteria.  A recent review by Sorek et al. brings one such example into focus.

One curious feature of bacterial genome is the occurrence of arrays of direct repeats in which the repeated units are separated by so-called spacers of unique sequence unrelated to the repeat units.  The sizes of the repeat units vary from bacteria to bacteria, ranging from between 24 to 47 bp.  Likewise, the spacer sizes vary from 26-72 bp.  These arrays are flanked by an apparent leader sequence, and yet again by arrays of protein-coding (CAS) genes, the number and composition of which vary considerably from bacteria to bacteria.  The general arrangement is shown in the following figure, which is part a of Figure 1 from Sorek et al. (shown beneath the fold):